All eukaryotic cells use multiple mitogen-activated protein kinase (MAPK) cascades to respond to many external stimuli that regulate proliferation, differentiation, survival and response to stress. MAPK cascades can function both downstream and upstream of oncoproteins and anti-oncoproteins that regulate cell survival. Thus, understanding basic mechanisms involved in MAPK cascade activation and the maintenance of pathway specificity is of general relevance and importance. The emerging theme of signal transduction through MAPK cascades is that they often use scaffold/adapter proteins to form higher order molecular assemblies within cells, with spacial organization playing a critical role. Spatial integrity is critical during the initial activation step of receptor-mediated signal transduction pathways, when cytoplasmic components such as scaffolds, kinases or other enzymes must be physically linked to membrane receptors and G proteins that sense the stimuli. The broad goals of this proposal are to understand the molecular mechanism of activation of a MAPK cascade, with an emphasis on the steps that lead to the assembly of an active signaling complex at the cell cortex. The mating pathway of S. cerevisiae is an excellent model system to study the mechanism of activation of a conserved receptor/G protein-coupled MAP kinase cascade. The Ste5 scaffold is a key specificity determinant for this pathway. Ste5 tethers the MAPKKKK Ste11, MAPKK Ste7 and MAPK Fus3 into a complex and is recruited to the Gbeta subunit of the G protein in the presence of stimulus. This recruitment event is essential for activation of Ste11 by Ste20, a PAK-like kinase that binds both Cdc42 GTPase and Gbeta at the cell cortex. The recruitment of Ste5 to Gbeta involves a novel localization pathway in which Ste5 first shuttles through the nucleus. We have identified a number of regulators of this localization pathway. Several of the regulators of nuclear shuttling and recruitment are conserved components of the cytoskeleton, suggesting that the link between nuclear shuttling and recruitment may be generally relevant.
The Specific Aims of this proposal are to 1) Define the cis-elements of Ste5 that regulate its localization, 2) Analyze regulators of nuclear import of Ste5, 3) Analyze regulators of nuclear export of Ste5, 4) Define the dictates of an active Ste5 oligomer that permit nuclear shuttling, membrane recruitment and kinase association, 5) Define specificity determinants that ensure that the mating MAPK, Fus3, is only activated by mating pheromone.
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